RESUMEN
Ageritin is the first reported ribotoxin-like protein from basidiomycetes fungi. It can induce ribosomal integrity damage and translation block, and interferes with mitochondrial redox activity of some glioma and neuroblastoma cell lines. Herein, Ageritin has been investigated as a valuable neurotoxin towards either undifferentiated or retinoic acid (RA)-differentiated SH-SY5Y neuroblastoma cells showing a selective cell toxicity against undifferentiated cells. MTT and sulforhodamine B (SRB) assays highlighted that Ageritin markedly decreases the mitochondrial redox activity and viability of undifferentiated cells, meanwhile inducing evident morphological changes eliciting neuronal-like appearance in these cells. Data from lactate dehydrogenase release assay, cytofluorimetric analysis and caspase-3 enzymatic activity measurement suggest that Ageritin promotes cell death through a caspase-dependent apoptotic pathway. The Z-VAD-FMK caspase inhibitor was able to prevent this apoptotic pathway activation. Based on the interesting behaviour of Ageritin vs. SH-SY5Y cells, the development of a scale-up procedure to obtain the purified protein in larger amounts (yield 2.5 mg per 100 g) has been optimized.
Asunto(s)
Agaricales/química , Diferenciación Celular/efectos de los fármacos , Extractos Vegetales/farmacología , Ribonucleasas/farmacología , Apoptosis/efectos de los fármacos , Caspasa 3/genética , Caspasa 3/metabolismo , Línea Celular Tumoral , Humanos , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Ribonucleasas/química , Ribonucleasas/aislamiento & purificaciónRESUMEN
The aim of this review is to cover most recent research on plant pathogenesis- and defenserelated proteins from latex-bearing medicinal plant Chelidonium majus (Papaveraceae) in the context of its importance for latex activity, function, pharmacological activities, and antiviral medicinal use. These results are compared with other latex-bearing plant species and recent research on proteins and chemical compounds contained in their latex. This is the first review, which clearly summarizes pathogenesisrelated (PR) protein families in latex-bearing plants pointing into their possible functions. The possible antiviral function of the latex by naming the abundant proteins present therein is also emphasized. Finally latex-borne defense system is hypothesized to constitute a novel type of preformed immediate defense response against viral, but also non-viral pathogens, and herbivores.
Asunto(s)
Antivirales/química , Chelidonium/química , Látex/química , Proteínas de Plantas/química , Alcaloides/química , Alcaloides/aislamiento & purificación , Alcaloides/farmacología , Antivirales/aislamiento & purificación , Antivirales/farmacología , Bencilisoquinolinas/química , Bencilisoquinolinas/aislamiento & purificación , Bencilisoquinolinas/farmacología , Catecol Oxidasa/química , Catecol Oxidasa/aislamiento & purificación , Catecol Oxidasa/farmacología , Quitinasas/química , Quitinasas/aislamiento & purificación , Quitinasas/farmacología , Endopeptidasas/química , Endopeptidasas/aislamiento & purificación , Endopeptidasas/farmacología , Lipooxigenasa/química , Lipooxigenasa/aislamiento & purificación , Lipooxigenasa/farmacología , Peroxidasas/química , Peroxidasas/aislamiento & purificación , Peroxidasas/farmacología , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/farmacología , Ribonucleasas/química , Ribonucleasas/aislamiento & purificación , Ribonucleasas/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
In recent years, several studies have demonstrated that the RNASET2 gene is involved in the control of tumorigenicity in ovarian cancer cells. Furthermore, a role in establishing a functional cross-talk between cancer cells and the surrounding tumor microenvironment has been unveiled for this gene, based on its ability to act as an inducer of the innate immune response. Although several studies have reported on the molecular features of RNASET2, the details on the mechanisms by which this evolutionarily conserved ribonuclease regulates the immune system are still poorly defined. In the effort to clarify this aspect, we report here the effect of recombinant human RNASET2 injection and its role in regulating the innate immune response after bacterial challenge in an invertebrate model, the medicinal leech. We found that recombinant RNASET2 injection induces fibroplasias, connective tissue remodeling and the recruitment of numerous infiltrating cells expressing the specific macrophage markers CD68 and HmAIF1. The RNASET2-mediated chemotactic activity for macrophages has been further confirmed by using a consolidated experimental approach based on injection of the Matrigel biomatrice (MG) supplemented with recombinant RNASET2 in the leech body wall. One week after injection, a large number of CD68+ and HmAIF-1+ macrophages massively infiltrated MG sponges. Finally, in leeches challenged with lipopolysaccharides (LPS) or with the environmental bacteria pathogen Micrococcus nishinomiyaensis, numerous macrophages migrating to the site of inoculation expressed high levels of endogenous RNASET2. Taken together, these results suggest that RNASET2 is likely involved in the initial phase of the inflammatory response in leeches.
Asunto(s)
Tejido Conectivo/patología , Hirudo medicinalis/fisiología , Inflamación/patología , Proteínas Recombinantes/farmacología , Ribonucleasas/farmacología , Proteínas Supresoras de Tumor/farmacología , Fosfatasa Ácida/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Colágeno/metabolismo , Tejido Conectivo/efectos de los fármacos , Crioultramicrotomía , Combinación de Medicamentos , Pruebas de Enzimas , Técnica del Anticuerpo Fluorescente , Hirudo medicinalis/anatomía & histología , Hirudo medicinalis/efectos de los fármacos , Hirudo medicinalis/ultraestructura , Humanos , Laminina/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Proteoglicanos/metabolismoRESUMEN
A ribonuclease, with a molecular mass of 22.5 kDa and an N-terminal sequence exhibiting resemblance to previously isolated buckwheat storage proteins and allergens, was isolated from Japanese large brown buckwheat seeds. The ribonuclease was purified using a simple protocol that comprised ion exchange chromatography on Q-Sepharose and DEAE-cellulose and gel filtration on Superdex 75. The ribonuclease exhibited low activity toward poly U, lower activity toward poly C, and very low activity toward poly A and poly G. The enzyme was activated upon exposure to 10 mM of Fe(2+) and Zn(2+) ions but was inhibited by Ca(2+), Mg(2+), and Mn(2+) ions at the same concentration. The optimum pH and optimum temperature for the enzyme were pH 9 and 60 °C, respectively. It inhibited proliferation of HepG2 hepatoma and MCF 7 breast cancer cells, with an IC50 value of 79.2 and 63.8 µM, respectively. It potently inhibited HIV-1 reverse transcriptase activity with an IC50 of 48 µM. However, there were no antifungal and mitogenic activities.
Asunto(s)
Fagopyrum/enzimología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/enzimología , Proteínas de Plantas/farmacología , Ribonucleasas/farmacología , Secuencia de Aminoácidos , Línea Celular Tumoral , Activación Enzimática , Estabilidad de Enzimas , Fagopyrum/química , Fagopyrum/genética , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/metabolismo , VIH-1/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Peso Molecular , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Ribonucleasas/química , Ribonucleasas/aislamiento & purificación , Ribonucleasas/metabolismo , Semillas/química , Semillas/enzimología , Semillas/genéticaRESUMEN
Onconase, a member of the pancreatic ribonuclease A superfamily, is currently in Phase III clinical trials for treatment of unresectable malignant mesothelioma. The anticancer effect of onconase may relate to its intracellular target, a non-coding RNA. Some non-coding RNAs are aberrantly expressed in cancer cells. This discovery is creating new interest in drugs that target RNA. Conjugating onconase to agents that recognize tumor associated molecules further increases its potency and specificity. Analysis of onconase activity when directed to two different internalizing and one non-internalizing receptor reveals that the ideal targeting agents would rapidly enter lysosomal compartments before onconase escaped to the cytosol. Antibody-onconase conjugates are effective in preclinical models, cause little non-specific toxicities in mice and have favorable formulation properties. Understanding the reason for their potency coupled with understanding novel RNA-based mechanisms of tumor cell death will lead to improved variations of targeted onconase.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Endocitosis/efectos de los fármacos , Inmunoconjugados/uso terapéutico , Neoplasias/tratamiento farmacológico , Ribonucleasas/uso terapéutico , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Humanos , Inmunoconjugados/administración & dosificación , Inmunoconjugados/inmunología , Inmunoconjugados/farmacología , Pruebas de Sensibilidad Microbiana , Neoplasias/inmunología , Neoplasias/metabolismo , Receptores de Transferrina/inmunología , Ribonucleasas/administración & dosificación , Ribonucleasas/inmunología , Ribonucleasas/farmacología , Lectina 2 Similar a Ig de Unión al Ácido Siálico/inmunologíaAsunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Endorribonucleasas/farmacología , Activación de Linfocitos/efectos de los fármacos , Antineoplásicos/inmunología , Antineoplásicos/farmacología , Biomarcadores/metabolismo , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Células Cultivadas , Citocinas/inmunología , Evaluación Preclínica de Medicamentos , Endorribonucleasas/inmunología , Humanos , Activación de Linfocitos/inmunología , Ribonucleasas/inmunología , Ribonucleasas/farmacologíaRESUMEN
Bleomycin is a clinically used antitumor antibiotic long thought to function therapeutically at the level of DNA cleavage. Recently, it has become clear that bleomycin can also cleave selected members of all major classes of RNA. Using the computer program COMPARE to search the database established by the Anticancer Drug Screening Program of the National Cancer Institute, a possible mechanism-based correlation was found between onconase, an antitumor ribonuclease currently being evaluated in phase III clinical trials, and the chemotherapeutic agent bleomycin. Following these observations, experimentation revealed that bleomycin caused tRNA cleavage and DNA-independent protein synthesis inhibition in rabbit reticulocyte lysate and when microinjected into Xenopus oocytes. The correlation of protein synthesis inhibition to the previously reported site-specific RNA cleavage caused by bleomycin supports the thesis that RNA cleavage may constitute an important element of the mechanism of action of bleomycin.
Asunto(s)
Antineoplásicos/farmacología , Bleomicina/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , ARN/efectos de los fármacos , Animales , Línea Celular Tumoral , Sistema Libre de Células , Evaluación Preclínica de Medicamentos/métodos , Humanos , Oocitos , ARN/metabolismo , Conejos , Reticulocitos , Ribonucleasas/metabolismo , Ribonucleasas/farmacología , Programas Informáticos , XenopusRESUMEN
Onconase is an amphibian protein that is now in Phase III clinical trials as a cancer chemotherapeutic. Human pancreatic ribonuclease (RNase 1) is homologous to Onconase but is not cytotoxic. Here, ERDD RNase 1, which is the L86E/N88R/G89D/R91D variant of RNase 1, is shown to have conformational stability and ribonucleolytic activity similar to that of the wild-type enzyme but > 10(3)-fold less affinity for the endogenous cytosolic ribonuclease inhibitor protein. Most significantly, ERDD RNase 1 is toxic to human leukemia cells. The addition of a non-native disulfide bond to ERDD RNase 1 not only increases the conformational stability of the enzyme but also increases its cytotoxicity such that its IC(50) value is only 8-fold greater than that of Onconase. Thus, only a few amino acid substitutions are necessary to make a human protein toxic to human cancer cells. This finding has significant implications for human cancer chemotherapy.
Asunto(s)
Antineoplásicos/farmacología , Ribonucleasa Pancreática/química , Ribonucleasa Pancreática/farmacología , Ribonucleasa Pancreática/toxicidad , Ribonucleasas/farmacología , Secuencia de Aminoácidos , Aminoácidos/química , División Celular , Cisteína/química , ADN Complementario/metabolismo , Disulfuros , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Agar , Humanos , Concentración 50 Inhibidora , Células K562 , Cinética , Leucemia/tratamiento farmacológico , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Ribonucleasa Pancreática/metabolismo , Espectrometría de Fluorescencia , Temperatura , Células Tumorales CultivadasRESUMEN
An isolation procedure, consisting of ion exchange chromatography on CM-Sepharose, affinity chromatography on Affi-gel blue gel, and fast protein liquid chromatography on Mono S, was utilized to purify a base-nonspecific, heterodimeric ribonuclease (RNase) with diverse activities from roots of the sanchi ginseng Panax notoginseng. The RNase is unique in that it consists of two different nonglycoprotein subunits with a molecular weight of 27 and 29 kDa, respectively. The latter subunit is characterized by an N-terminal sequence showing remarkable similarity to that of the bitter gourd RNase. The Panax notoginseng RNase demonstrates potent RNase and translation-inhibitory activities. In addition, it exhibits antiproliferative activity toward leukemia L1210 cells and antifungal activity against Physalospora piricola and Coprinus comatus. Its RNase activity is not heat-resistant, unlike most RNases which are thermostable.
Asunto(s)
Antifúngicos/farmacología , Supervivencia Celular/efectos de los fármacos , Panax/enzimología , Plantas Medicinales , Ribonucleasas/química , Ribonucleasas/farmacología , Animales , Antifúngicos/química , Antifúngicos/aislamiento & purificación , División Celular/efectos de los fármacos , China , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Cromatografía Liquida , Coprinus/efectos de los fármacos , Coprinus/crecimiento & desarrollo , Dimerización , Estabilidad de Enzimas , Calor , Cinética , Leucemia L1210 , Ratones , Pruebas de Sensibilidad Microbiana , Peso Molecular , Raíces de Plantas/enzimología , Biosíntesis de Proteínas/efectos de los fármacos , Subunidades de Proteína , Conejos , Reticulocitos/metabolismo , Ribonucleasas/aislamiento & purificación , Ribonucleasas/metabolismo , Homología de Secuencia de Aminoácido , Termodinámica , Células Tumorales CultivadasRESUMEN
LL2, an anti-CD22 monoclonal antibody against B-cell lymphoma, was covalently linked to the amphibian ribonuclease, onconase, a member of the pancreatic RNase A superfamily. LL2 increased in vitro potency (10 000-fold) and specificity against human Daudi Burkitt lymphoma cells while decreasing systemic toxicity of onconase. Monensin further increased potency of LL2-onconase on Daudi cells (IC(50), 20 and 1.5 pM, absence and presence of monensin, respectively). A 1-hour exposure to LL2-onconase was sufficient to kill Daudi cells in culture. These favorable in vitro properties translated to significant antitumor activity against disseminated Daudi lymphoma in mice with severe combined immunodeficiency disease. In mice inoculated with tumor cells intraperitoneally (ip), LL2-onconase (100 microg 5 times ip every day) increased the life span of animals with minimal disease 200%. The life span of mice with advanced disseminated Daudi lymphoma (tumor cells inoculated intravenously) was increased 135%. Mice injected with LL2-onconase tolerated a dose as high as 300 mg/kg. Because both onconase and LL2 are in clinical trials as cancer therapeutics, the covalently linked agents should be considered for treatment of non-Hodgkin lymphoma.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Moléculas de Adhesión Celular , Lectinas , Ribonucleasas/farmacología , Animales , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales/toxicidad , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos B/inmunología , Antineoplásicos/química , Antineoplásicos/toxicidad , Muerte Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Estabilidad de Medicamentos , Femenino , Humanos , Inmunotoxinas/farmacología , Inmunotoxinas/uso terapéutico , Inmunotoxinas/toxicidad , Cinética , Linfoma no Hodgkin/tratamiento farmacológico , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Modelos Animales , Trasplante de Neoplasias , Páncreas/enzimología , Ribonucleasas/uso terapéutico , Ribonucleasas/toxicidad , Lectina 2 Similar a Ig de Unión al Ácido Siálico , Tasa de Supervivencia , Células Tumorales CultivadasRESUMEN
The seeds of Lagenaria siceraria (Family Cucurbitaceae) were extracted with water and the extract was lyophilized. The lyophilized extract was chromatographed on a DEAE-cellulose column in 10 mM Tris-HCl buffer (pH 7.2). The unadsorbed fraction was applied to an Affi-gel Blue gel column previously equilibrated with the same buffer. After removal of unadsorbed materials, the adsorbed proteins were eluted with 1.5 M NaCl in the Tris-HCl buffer. After dialysis the adsorbed fraction was loaded on a CM-Sepharose CL-6B column which had been equilibrated with and was eluted with the same buffer. After elution of unadsorbed proteins, the column was eluted with a gradient of 0-1 M NaCl in 10 mM Tris-HCl buffer (pH 7.2). The fraction eluting at about 0.55 M NaCl, which represented pure ribosome inactivating protein (RIP), inhibited cell-free translation in a rabbit reticulocyte system with an IC50 of 0.21 nM and exerted ribonuclease activity on yeast tRNA with an activity of 45 U/mg. The RIP was designated lagenin. It possessed a molecular weight of 20 kDa, smaller than the range of 26-32 kDa reported for other RIPs. The N-terminal sequence of lagenin exhibited a lesser extent of similarity to those of other Cucurbitaceae RIPs, characterized by a deletion of the first three amino acid residues and a replacement of the 4th (Phe), 17th (Phe), 18th (Ile) and 22nd (Arg) residues which are invariant in other RIPs.
Asunto(s)
Cucurbitaceae/química , Medicamentos Herbarios Chinos/química , Extractos Vegetales/farmacología , Proteínas de Plantas/farmacología , Ribonucleasas/antagonistas & inhibidores , Ribonucleasas/farmacología , Ribosomas/efectos de los fármacos , Semillas/química , Secuencia de Aminoácidos , Animales , Cromatografía DEAE-Celulosa , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Peso Molecular , Extractos Vegetales/aislamiento & purificación , Proteínas de Plantas/aislamiento & purificación , Conejos , Ribonucleasas/aislamiento & purificaciónRESUMEN
We have identified and characterized protein factors from mung bean (Vigna radiata) nuclear extracts that specifically bind the single-stranded G-rich telomeric DNA repeats. Nuclear extracts were prepared from three different types of plant tissue, radicle, hypocotyl, and root, in order to examine changes in the expression patterns of telomere-binding proteins during the development of mung bean. At least three types of specific complexes (A, B, and C) were detected by gel retardation assays with synthetic telomere and nuclear extract from radicle tissue, whereas the two major faster-migrating complexes (A and B) were formed with nuclear extracts from hypocotyl and root tissues. Gel retardation assays also revealed differences in relative amount of each complex forming activity in radicle, hypocotyl, and root nuclear extracts. These data suggest that the expression of telomere-binding proteins is developmentally regulated in plants, and that the factor involved in the formation of complex C may be required during the early stages of development. The binding factors have properties of proteins and are hence designated as mung bean G-rich telomere-binding proteins (MGBP). MGBPs bind DNA substrates with three or more single-stranded TTTAGGG repeats, while none of them show binding affinity to either double-stranded or single-stranded C-rich telomeric DNA. These proteins have a lower affinity to human telomeric sequences than to plant telomeric sequences and do not exhibit a significant binding activity to Tetrahymena telomeric sequence or mutated plant telomeric sequences, indicating that their binding activities are specific to plant telomere. Furthermore, RNase treatment of the nuclear extracts did not affect the complex formation activities. This result indicates that the single-stranded telomere-binding activities may be attributed to a simple protein but not a ribonucleoprotein. The ability of MGBPs to bind specifically the single-stranded TTTAGGG repeats may suggest their in vivo functions in the chromosome ends of plants.
Asunto(s)
ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Fabaceae/metabolismo , Plantas Medicinales , Unión Competitiva/efectos de los fármacos , Núcleo Celular/metabolismo , Cesio/farmacología , Cloruros/farmacología , Proteínas de Unión al ADN/genética , Electroforesis en Gel de Poliacrilamida , Fabaceae/genética , Fabaceae/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Calor , Hipocótilo/metabolismo , Proteínas Nucleares/metabolismo , Oligonucleótidos/metabolismo , Unión Proteica/efectos de los fármacos , Secuencias Repetitivas de Ácidos Nucleicos , Ribonucleasas/metabolismo , Ribonucleasas/farmacología , Semillas/crecimiento & desarrollo , Semillas/metabolismo , Cloruro de Sodio/farmacología , Telómero/genética , Telómero/metabolismoRESUMEN
Type I protein arginine N-methyltransferases catalyze the formation of omega-NG-monomethylarginine and asymmetric omega-NG, NG-dimethylarginine residues using S-adenosyl-l-methionine as the methyl donor. In vitro these enzymes can modify a number of soluble methyl-accepting substrates in yeast and mammalian cell extracts including several species that interact with RNA. We treated normal and hypomethylated Saccharomyces cerevisiae and RAT1 cell extracts with RNase prior to in vitro methylation by recombinant protein N-arginine methyltransferases and found that the methylation of certain polypeptides is enhanced up to 12-fold whereas that of others is diminished. 2-D gel electrophoresis of RNase-treated yeast extracts allowed us to tentatively identify the glycine- and arginine-rich (GAR) domain-containing proteins Gar1, Nop1, Sbp1, and Npl3 as major methyl-acceptors based on their known isoelectric points and apparent molecular weights. These results suggest that the methylation and RNA-binding of GAR domain-containing proteins in vivo may regulate protein-nucleic acid or protein-protein interactions.
Asunto(s)
Proteínas de Peces , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas de Unión al ARN , Ribonucleasas/farmacología , Ribonucleoproteínas Nucleolares Pequeñas , Proteínas de Saccharomyces cerevisiae , Animales , Proteínas Sanguíneas/análisis , Línea Celular , Proteínas Fúngicas/análisis , Metilación , Proteínas Nucleares/análisis , Péptidos/análisis , Péptidos/metabolismo , Ratas , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Especificidad por SustratoRESUMEN
A polymerase chain reaction product was used to isolate mouse brain cDNA clones coding for isoforms of the beta subunit of voltage-dependent Ca2+ channels. The two mouse brain beta 2 subunit cDNA clones described, beta 2a and beta 2b, differed by alternative splicing within the coding region but possessed a unique amino terminus not yet reported in other beta 2 subunit cDNAs. Northern blot and RNase protection analyses demonstrated that both mRNA isoforms could be detected in highest abundance in heart and brain and at lower levels in lung, kidney, and testis. In a novel assay for beta 2 subunit function, COS-1 cells were transfected with alpha 1 and beta 2 subunit expression vectors and assayed for increases in intracellular Ca2+ concentration by using fura-2 imaging. Co-transfection of COS-1 cells with the mouse brain class C-1 alpha 1 subunit expression vector and either of the beta 2 subunit expression vectors resulted in increases in intracellular Ca2+ concentration after stimulation with elevated K+ and the dihydropyridine agonist Bay K 8644. Transfection of either alpha 1 or beta 2 subunit expression vectors alone did not result in an elevation of intracellular Ca2+ concentration. Electrophysiological recording of human embryonic kidney 293 cells transfected with the expression vector for the alpha 1 subunit alone or with either beta 2 subunit demonstrated expression of voltage-dependent Ca2+ channels that were dihydropyridine sensitive. Currents formed by expression of only the alpha 1 subunit were small and slowly inactivated. In contrast, the currents formed by coexpression of alpha 1 subunits with either beta 2 subunit were larger and inactivated more rapidly. Dihydropyridine binding studies demonstrated that coexpression of alpha 1 subunits with beta 2 subunits increased the density of functional receptors, compared with expression of alpha 1 subunits alone. These experiments suggested that coexpression of the alpha 1 and beta 2 subunits produced functional dihydropyridine-sensitive Ca2+ channels and that both beta subunit isoforms had modulatory effects on these channels.
Asunto(s)
Canales de Calcio/análisis , Calcio/metabolismo , Fura-2 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Canales de Calcio/genética , Línea Celular , ADN Complementario/aislamiento & purificación , Isradipino/metabolismo , Ratones , Datos de Secuencia Molecular , Conejos , Ribonucleasas/farmacología , TransfecciónAsunto(s)
Antiinflamatorios/uso terapéutico , Estomatitis/tratamiento farmacológico , Enfermedad Aguda , Antiinflamatorios/efectos adversos , Antiinflamatorios/farmacología , Enfermedad Crónica , Desoxirribonucleasas/farmacología , Desoxirribonucleasas/uso terapéutico , Humanos , Péptido Hidrolasas/efectos adversos , Péptido Hidrolasas/farmacología , Péptido Hidrolasas/uso terapéutico , Extractos Vegetales/efectos adversos , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Ribonucleasas/farmacología , Ribonucleasas/uso terapéutico , Factores de TiempoRESUMEN
The antiviral activity of a bacterial ribonuclease conjugate with chitosane of Kamchatka crab (in a form of water soluble chito-oligosaccharides) has been studied. The conjugate inhibitory activity for A and B viruses as well as to Sindbis arbovirus in tissue cultures is shown. The preparation efficiency at intramuscular and intranasal administration was observed at experimental influenza infection of white mice.
Asunto(s)
Antivirales/farmacología , Ribonucleasas/farmacología , Animales , Antivirales/uso terapéutico , Bacillus/enzimología , Braquiuros/enzimología , Quitina/análogos & derivados , Quitina/farmacología , Quitina/uso terapéutico , Quitosano , Combinación de Medicamentos , Evaluación Preclínica de Medicamentos , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza B/efectos de los fármacos , Ratones , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Ribonucleasas/uso terapéutico , Virus Sindbis/efectos de los fármacosRESUMEN
The enzyme complex delta 5-3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta HSD) is involved in the biosynthesis of all classes of steroids, namely glucocorticoids, mineralocorticoids, progesterone, and sex steroids. To obtain information on the precise localization of 3 beta HSD in rat gonads and adrenal glands, two complementary cytochemical techniques were used; immunocytochemical localization was achieved with antibodies developed against purified human placental 3 beta HSD, while 3 beta HSD mRNA localization was achieved by in situ hybridization performed with a recently cloned rat 3 beta HSD cDNA. In the testis, specific immunostaining was restricted to the cytoplasm of the interstitial cells, while by in situ hybridization, specific silver grains were also seen over the interstitial cells. In the ovary, immunostaining was found in the cytoplasm of cells of the corpus luteum and theca interna, while the granulosa cells of the follicles showed no positive reaction. By in situ hybridization, a specific hybridization signal was observed over granulosa cells of the corpus luteum, which are mainly responsible for progesterone secretion, and to a lesser extent over theca interna cells, known for their role in secreting C19 androgens. In the adrenals, the three zones of the cortex were equally immunolabeled, whereas no staining could be detected in the medulla. Similarly, by in situ hybridization, silver grains were located over the zona glomerulosa, fasciculata, and reticularis, while no specific autoradiographic reaction could be observed on the chromaffin cells of the medulla. The present study provides new information about the precise cellular localization of 3 beta HSD in the adrenal glands and gonads in the rat, thus providing useful information about the site of action of 3 beta HSD, especially in the gonads. Moreover, the approaches used for localization studies, especially quantitative in situ hybridization, should provide a useful tool for assessing the role of hormones on 3 beta HSD expression in the different compartments of the gonads and adrenal glands.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/análisis , Glándulas Suprarrenales/enzimología , Isomerasas/análisis , Complejos Multienzimáticos/análisis , Ovario/enzimología , Progesterona Reductasa/análisis , Esteroide Isomerasas/análisis , Testículo/enzimología , Animales , Autorradiografía , Cuerpo Lúteo/enzimología , Citoplasma/enzimología , Sondas de ADN , Femenino , Inmunohistoquímica , Células Intersticiales del Testículo/enzimología , Masculino , Hibridación de Ácido Nucleico , Folículo Ovárico/enzimología , Ratas , Ratas Endogámicas , Ribonucleasas/farmacología , Células Tecales/enzimología , Distribución TisularRESUMEN
A biotinylated antisense oligonucleotide probe specific for the glycopeptide sequence of arginine vasopressin mRNA has been used with amplified detection for visualisation of arginine vasopressin mRNA in the rat hypothalamus. RNAase pretreatment to destroy arginine vasopressin mRNA and use of excess complementary oligonucleotide (sense) to absorb the biotinylated antisense oligonucleotide demonstrated the reaction is specific for arginine vasopressin mRNA. Further, dehydration of rats using 2% saline resulted in an increase in specific staining. The staining is localized to those neurones in the hypothalamus known to contain arginine vasopressin.
Asunto(s)
Arginina Vasopresina/metabolismo , Biotina , ADN , Hipotálamo/metabolismo , Hibridación de Ácido Nucleico , ARN Mensajero/análisis , Animales , Inmunohistoquímica/métodos , Masculino , Sondas de Oligonucleótidos , Ratas , Ratas Endogámicas , Ribonucleasas/farmacologíaRESUMEN
The capability of dietary selenium (Se) to augment the immune response was evaluated in 96 crossbred weanling swine. Six groups of 16 pigs were fed diets with Se supplemented as sodium selenite at 0, 0.3, 0.6, 0.9, 1.2, and 1.5 mg/kg. The basal diet contained 0.068 mg of Se/kg. Weight gain, feed consumption, and feed efficiency were similar for all diets. Whole blood concentrations of Se linearly increased as the dietary concentrations of Se increased. The humoral response was monitored by immunoglobulin G titers to lysozyme and ribonuclease, using an enzyme-linked immunosorbent assay. Although no significant difference in immunoglobulin G titers to either antigen was detected among diets, a similar trend in antibody response was noted. The diet with 0.9 mg of added Se/kg produced the highest antibody response to both antigens, whereas the diet with 0.3 mg of added Se/kg produced the lowest titers for both antigens. Cell-mediated immunity was evaluated in the pigs by the dermal response to phytohemagglutinin. Significant difference was not detected in pigs fed the various diets in terms of the mean diameters of their dermal reactions to phytohemagglutinin injections. Although blood concentrations of Se were increased, rate and efficiency of weight gain and humoral and cell-mediated immunity were not significantly improved by adding 0.3 to 1.5 mg of Se/kg to diets.